Alphaviruses comprise a set of genetically, structurally, and serologically related arthropod-borne viruses of the Togaviridae family. Twenty-six known viruses and virus subtypes have been classified within the alphavirus genus, including, Sindbis virus (SIN), Semliki Forest virus (SFV), Ross River virus (RRV), and Venezuelan equine encephalitis virus (VEE).
Several members of the alphavirus genus are being developed as “replicon” expression vectors for use as vaccines and therapeutics. Replicon vectors may be utilized in several formats, including DNA, RNA, and to make recombinant virus-like particles containing the replicon vectors (replicon particles). Such replicon vectors have been derived from alphaviruses that include, for example, SIN (Xiong et al. (1989) Science 243:1188-1191; Dubensky et al., (1996) J. Virol. 70:508-519; Hariharan et al. (1998) J. Virol. 72:950-958; Polo et al. (1999)PNAS 96:4598-4603), Semliki Forest virus (Liljestrom (1991) Bio/Technology 9:1356-1361; Berglund et al. (1998) Nat. Biotech. 16:562-565), and VEE (Pushko et al. (1997) Virology 239:389-401). A wide body of literature has now demonstrated efficacy of using alphavirus replicon vectors for applications such as vaccines (see for example, Dubensky et al., ibid; Berglund et al., ibid; Hariharan et al., ibid, Pushko et al., ibid; Polo et al., ibid; Davis et al. (2000) J Virol. 74:371-378; Schlesinger & Dubensky (1999) Curr Opin. Biotechnol. 10:434-439; Berglund et al. (1999) Vaccine 17:497-507).
The use of alphavirus replicon vectors as nucleic acid-based vaccines may provide certain advantages as compared to other nucleic acid expression vectors. Through the years, several terms including alphavirus vector, alphavirus vector construct, alphavirus replicon, alphavirus RNA replicon, alphavirus vector replicon, Eukaryotic Layered Vector Initiation System (ELVIS), alphavirus plasmid replicon and the like have emerged to describe alphavirus replicon vectors.
In addition to their use as gene delivery vehicles, alphavirus replicon vectors have also been described for use to generate recombinant viral or virus-like particles (replicon particles), which are themselves useful in prophylactic and therapeutic applications. See, e.g., Polo et al. (1999) Proc Natl Acad Sci USA 96(8):4598-4603. Alphavirus replicon particles are typically generated using a multi-component system that separates the various elements required for particle formation. Separation of the various elements reduces the risk of generating undesirable, replication-competent virus. The systems typically include: 1) a replicon vector, which contains elements necessary for its own intracellular replication (e.g., nonstructural protein coding sequences) but lacks one or more structural protein encoding elements needed for production of progeny particles, and 2) one or more structural protein expression cassette constructs (e.g., defective helpers) that encode alphavirus structural proteins (e.g., capsid, glycoproteins) required for packaging. The replicon constructs and the defective helper constructs can be introduced directly into cells as RNAs or launched from DNA in either transiently or stably transfected cells (e.g., packaging cell lines or PCL). See, e.g., Polo et al. (1999) Proc. Nat'l Acad. Sci USA 96:4598-4603; U.S. Pat. Nos. 6,465,634; 6,426,196; 6,376,236; 6,342,372; 6,015,686; and 5,843,723. Dubensky, T W et al. (1996) J. Virology 70(1):508-519; Frolov et al. (1996). Proc Natl Acad Sci USA. 93(21):11371-11377).
Ideally, the populations of alphavirus replicon particles used in prophylactic or therapeutic applications would be substantially homogenous and contain only the replicon RNA. However, even when the packaging elements are separated, particles containing additional RNA species (e.g., defective helper RNA) can occur. This undesirable packaging of non-replicon RNA species is also termed “co-packaging.”
Thus, despite the advances in alphaviral vector technology, there remains a need for pharmaceutical compositions comprising and methods of making and using alphaviral vectors and alphavirus replicon particles, for example to reduce co-packaging.